The overarching goal of this Program is to understand the molecular mechanisms that regulate cell activation. Activation of lymphocytes through specific recognition of antigen poses narrowly balanced benefits and risks, and hence is subject to tight regulation. We know that some signals serve to modulate or terminate activation, while other signals induce cell unresponsiveness or death. The molecular mechanisms underlying the regulatory processes with different outcomes are unknown and largely undefined. The Program consists of five highly interactive projects involving six investigators, from five different departments of the University. These investigators bring innovative technology and incisive ideas to bear on the problem of lymphocyte activation and its regulation. The five projects cover a range of stimuli, cues and outcomes that result in various states of T cell activation or unresponsiveness, from the initial events of antigen recognition to the regulation of transcription. The projects cover a biological scale from nano-scale molecular interactions to whole cell and animal models. The program addresses the following areas: 1) Influence of TCR spatial organization on T cell responses, 2) Altered molecular architecture at the Immunological Synapse and T cell anergy, 3) Sprouty 1 as a novel inhibitor of T cell activation, 4) Regulation of calcium signaling in T cells by TFII-I, 5) Regulation of NF-kB by TCR and costimulatory signaling. These five projects support the overall goal of understanding the molecular mechanisms regulating cell activation. The program has developed out of years of interaction among a core group of investigators who in the last two years have been joined by new colleagues. Interactions between investigators are flourishing. The extent of synergy between group members is apparent from the detailed project descriptions. We want to understand the mechanisms of T cell responses to antigen. Understanding these mechanisms will lead to ways of turning up, or turning off immune responses that are helpful or harmful. PROJECT 1: The Influence of TCR spatial organization on T cell responses (PI [Schneck, Jonathan]) PROJECT 1 DESCRIPTION (provided by applicant): There is growing evidence that the clustering and spatial pattern of T cell receptors is critical for T cell responses. Changes in clustering and pattern can alter cell responsiveness from low to high, or even to unresponsive. We aim to investigate the genetics and the biophysics of T cell receptor clustering and pattern. Genetic screens will be used to identify pathways that are important for the enhanced clustering of T cell receptors found on activated T cells. We will use an shRNA screen to focus on pathways known to affect T cell receptor clustering -glycosylation and the role of cholesterol and its interaction with the cytoskeleton. Second we will analyze the impact of genes identified in the screen in binding of a TCR ligand, soluble peptide-MHC-lg complexes to T cells and in vitro and in vivo T cell responses. In addition we will engineer quantum dots that will present TCR ligands, MHC/peptide or anti-CD3 to naive and activated T cells. The quantum dots will be used to probe the lateral organization of TCR on naive and activated T cells. Blinking of bound quantum dots will be analyzed quantitatively to follow changes in receptor organization with time. Synergies with other projects: A specific aim of Project 2, is to analyze the TCR organization and SMAC formation in activated CD4 cells. Project 1's work on the genetic control of TCR clustering will be directly relevant to that project. Also, the nanoprobes and methods for characterizing TCR organization of this project will be used to probe the nano-organization of TCR on naive or activated CD4+ cells. In Project 3 quantum dots will be used to characterize changes in TCR clustering during induction of anergy in vivo. Quantum dots and genetic manipulation will also be used in collaboration with Dr. Desiderio's Project 4 to characterize T cell receptor states in cells and clones lacking TFII-I or carrying a mutated form of the protein. There has already been strong interaction with Project 5 in designing selection/screening assays for genetic control of TCR clustering. In addition using the techniques developed in Specific Aim 1 we will analyze the effects of the genes identified in Project 5 on TCR clustering. Many immune responses, for example responses to parasites, to tumors and to some viruses, are often blunted because T cells respond to antigen by turning off, rather than by activating and attacking their targets. Understanding the arrangement of T cell antigen receptors that is most likely to trigger T cells, will lead to development of methods to manipulate the T cell surface to produce a strong, focused response, or, to suppress response, rearranging the receptors to limit autoimmune responses.